The epidermal growth factor receptor (EGFR, HER1, erbB1), HER2/neu (erbB2), HER3 (erbB3), and HER4 (erbB4) comprise a family of homologous and interactive transmembrane receptor tyrosine kinases. Abundant evidence support the role of this signaling network in mammary gland development, transformation, and tumor progression. Recent studies indicate that inhibition of HER2 with the humanized IgG1 Herceptin induces tumor regressions and may alter the natural history of late metastatic breast cancers. However, the majority of patients with HER2-amplified breast cancers do not respond to Herceptin, implying that HER2 might be dispensable in most advanced cancers and supporting the presence of de novo or acquired mechanisms of resistance to HER2 inhibitors. During the previous funding period, we have reported that inhibition of phosphatidylinositol-3 kinase (PI3K) and its target the serine/threonine kinase Akt is a required step for the cell cycle arrest and/or apoptosis that occurs in HER2-dependent tumors once HER2 function is blocked. Inhibition of Akt relieves the Cdk inhibitor p27 Kip1 from phosphorylation at Thr 157 within its Akt consensus, resulting in redirection of p27 to the nucleus where it associates with and inhibits cyclin E/Cdk2 complexes, thus inducing growth arrest. Antisense p27 prevents the antitumor effect of HER2 inhibitors, suggesting that modulation of p27 levels and/or its localization also play a role in the growth arrest that follows HER2 blockade. We propose to test the hypothesis that amplification of the PI3K/Akt signaling pathway (in Herceptin-resistant cells) and low levels of p27 (in p27-haploinsufficient tumors) are mechanisms for resistance to Herceptin and/or HER2 kinase inhibitors. Defining those mechanisms of cellular resistance will identify potentially novel therapeutic targets and lead to the development of molecular approaches that can be combined with currently used inhibitors of the HER2 network. To achieve these goals, we will pursue the following specific aims: Specific Aim 1: To determine if amplification of the PI3K/Akt signaling pathway mediates acquired resistance to Herceptin in HER2-overexpressing BT-474 human breast cancer cells. Specific Aim 2: To identify by mass spectrometry changes in proteins and HER receptor-protein signaling complexes associated with resistance to EGFR/HER2 inhibitors in human breast cancer cells. Specific Aim 3. To determine if a threshold level of p27 is required for the antitumor action of Herceptin or HER network signaling inhibitors like OSI-774 in HER2-overexpressing transgenic tumors.